CD8+ T cells mediate recovery and immunopathology in West Nile virus encephalitis

Abstract

C57BL/6J mice infected intravenously with the Sarafend strain of West Nile virus (WNV) develop a characteristic central nervous system (CNS) disease, including an acute inflammatory reaction. Dose response studies indicate two distinct kinetics of mortality. At high doses of infection (10(8) PFU), direct infection of the brain occurred within 24 h, resulting in 100% mortality with a 6-day mean survival time (MST), and there was minimal destruction of neural tissue. A low dose (10(3) PFU) of infection resulted in 27% mortality (MST, 11 days), and virus could be detected in the CNS 7 days postinfection (p.i.). Virus was present in the hypogastric lymph nodes and spleens at days 4 to 7 p.i. Histology of the brains revealed neuronal degeneration and inflammation within leptomeninges and brain parenchyma. Inflammatory cell infiltration was detectable in brains from day 4 p.i. onward in the high-dose group and from day 7 p.i. in the low-dose group, with the severity of infiltration increasing over time. The cellular infiltrates in brain consisted predominantly of CD8(+), but not CD4(+), T cells. CD8(+) T cells in the brain and the spleen expressed the activation markers CD69 early and expressed CD25 at later time points. CD8(+) T-cell-deficient mice infected with 10(3) PFU of WNV showed increased mortalities but prolonged MST and early infection of the CNS compared to wild-type mice. Using high doses of virus in CD8-deficient mice leads to increased survival. These results provide evidence that CD8(+) T cells are involved in both recovery and immunopathology in WNV infection.

Figures

FIG. 1.

Histology of WNV-infected brains. Representative photomicrographs of brain sections from control and 103 PFU WNV-inoculated B6 mice. (A) Uninfected mouse brain; (B) vascular congestion and perivascular leukocyte infiltration in brain parenchyma, 7 days p.i.; (C) mild vacuolization and inflammation in brain parenchyma, 9 days p.i.; (D) severe vacuolization and inflammation in brain parenchyma, 11 days p.i. Neurons show degenerative changes with cytoplasmic rarefaction and rounding (arrows, panel C) and necrosis with nuclear compaction (arrow head, panel D). Panels A through D depict Hematoxylin-eosin stain, ×200 magnification. (E) Normal myelin display from uninfected mouse brain (blue stained); (F) vascular degeneration occurred mainly in the myelin area of the infected mouse brain. LFB stain, ×400 magnification.

FIG. 2.

IHC of WNV-infected brains. CD8+ cells localized in the brains of uninfected and 103 PFU WNV-infected B6 wt mice. (A) Undetectable CD8+ cells in the brains of uninfected mice; (B) perivascular CD8+ cell infiltration in brain parenchyma, 7 days p.i.; (C) vascular congestion and CD8+ cell (arrows) infiltration, 7 days p.i.; (D) perivascular CD8+ cell inflammation and infiltration of brain parenchyma, 9 days p.i.; (E) severe vascular congestion, perivascular and leptomeningeal CD8+ cell inflammation, and infiltration of leptomeninges and brain parenchyma, 11 days p.i.; (F) widespread CD8+ cell infiltration of brain parenchyma, 11 days p.i. Magnification, ×400.

FIG. 3.

Leukocyte isolation from WNV-infected mouse brains. Yields of inflammatory T cells in brains of WNV-infected mice. Mice were infected i.v. with a high dose (108 PFU) (A and B) or a low dose (103 PFU) (C and D) of virus. The infiltrated lymphocytes were isolated from the brains of individual mice by density gradient isolation at each time point and were analyzed using flow cytometry as described in Materials and Methods. T cells were undetectable in the brains of uninfected mice.

FIG. 4.

T-cell composition of spleens after WNV infection. Percentages of CD8+ (shaded bars) and CD4+ (empty bars) cells in spleens of mice infected with 108 PFU (upper panel) and 103 PFU (lower panel) of WNV. Values shown are the mean values ± standard errors of the means of three to five mice per group. The CD8/CD4 ratios are indicated by open diamonds (◊). *, significant difference (P < 0.05) between infected and control mice.

FIG. 5.

Comparison of virus spread in brain tissue from B6 and β2-m−/− mice on day 9 after 103 PFU of WNV infection i.v. Brain sections from B6 (A and B) and β2-m−/− (C and D) mice were stained for the WNV (right panel) and counterstained with hematoxylin (left panel) as described in Materials and Methods. Arrows denote individual infected neurons. Magnification, ×630.